This invention relates to materials and methods used for the generation of high titers of viral vectors, particularly recombinant adeno-associated virus (AAV) vectors. More specifically, the invention relates to AAV split-packaging genes, and cell lines comprising such genes, for use in the production of high titers of replication-incompetent AAV vectors.
Vectors based on adeno-associated virus (AAV) are believed to have utility for gene therapy but a significant obstacle has been the difficulty in generating such vectors in amounts that would be clinically useful for human gene therapy applications. This is a particular problem for in vivo applications such as direct delivery to the lung. Another important goal in the gene therapy context, discussed in more detail herein, is the production of vector preparations that are essentially free of replication-competent virions. The following description briefly summarizes studies involving adeno-associated virus and AAV vectors, and then describes a number of novel improvements according to the present invention that are useful for efficiently generating high titer recombinant AAV vector (rAAV) preparations suitable for use in gene therapy.
Adeno-associated virus is a defective parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General reviews of AAV may be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. I, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Examples of co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. The nature of the helper function is not entirely known but it appears that the helper virus indirectly renders the cell permissive for AAV replication. This belief is supported by the observation that AAV replication may occur at low efficiency in the absence of helper virus co-infection if the cells are treated with agents that are either genotoxic or that disrupt the cell cycle.
Although AAV may replicate to a limited extent in the absence of helper virus in these unusual conditions more generally infection of cells with AAV in the absence of helper functions results in the proviral AAV genome integrating into the host cell genome. If these cells are superinfected with a helper virus such as adenovirus, the integrated AAV genome can be rescued and replicated to yield a burst of infectious progeny AAV particles. The fact that integration of AAV appears to be efficient suggests that AAV would be a useful vector for introducing genes into cells for uses such as human gene therapy.
AAV has a very broad host range without any obvious species or tissue specificity and can replicate in virtually any cell line of human, simian or rodent origin provided that an appropriate helper is present. AAV is also relatively ubiquitous and has been isolated from a wide variety of animal species including most mammalian and several avian species.
AAV is not associated with the cause of any disease. Nor is AAV a transforming or oncogenic virus, and integration of AAV into the genetic material of human cells generally does not cause significant alteration of the growth properties or morphological characteristics of the host cells. These properties of AAV also recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application, such as retroviruses, adenoviruses, herpesviruses, or poxviruses, are disease-causing.
AAV particles are comprised of a proteinaceous capsid having three capsid proteins, VP1, VP2 and VP3, which enclose a DNA genome. The AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5xc3x97106 daltons and a length of approximately 4680 nucleotides. Individual particles package only one DNA molecule strand, but this may be either the xe2x80x9cplusxe2x80x9d or xe2x80x9cminusxe2x80x9d strand. Particles containing either strand are infectious and replication occurs by conversion of the parental infecting single strand to a duplex form and subsequent amplification of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids. Duplex or single-strand copies of AAV genomes can be inserted into bacterial plasmids or phagemids and transfected into adenovirus-infected cells; these techniques have facilitated the study of AAV genetics and the development of AAV vectors.
The AAV genome, which encodes proteins mediating replication and encapsidation of the viral DNA, is generally flanked by two copies of inverted terminal repeats (ITRs). In the case of AAV2, for example, the ITRs are each 145 nucleotides in length, flanking a unique sequence region of about 4470 nucleotides that contains two main open reading frames for the rep and cap genes (Srivastava et al., 1983, J. Virol., 45:555-564; Hermonat et al., J. Virol.51:329-339; Tratschin et al., 1984a, J. Virol., 51:611-619). The AAV2 unique region contains three transcription promoters p5, p19, and p40 (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA, 76:5567-5571) that are used to express the rep and cap genes. The ITR sequences are required in cis and are sufficient to provide a functional origin of replication (ori), signals required for integration into the cell genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. It has also been shown that the ITR can function directly as a transcription promoter in an AAV vector (Flotte et al., 1993, supra).
The rep and cap gene products are required in trans to provide functions for replication and encapsidation of viral genome, respectively. The rep gene is expressed from two promoters, p5 and p19, and produces four proteins. Transcription from p5 yields an unspliced 4.2 kb mRNA encoding a first Rep protein (Rep78), and a spliced 3.9 kb mRNA encoding a second Rep protein (Rep68). Transcription from p19 yields an unspliced mRNA encoding a third Rep protein (Rep52), and a spliced 3.3 kb mRNA encoding a fourth Rep protein (Rep40). Thus, the four Rep proteins all comprise a common internal region sequence but differ in their amino and carboxyl terminal regions. Only the large Rep proteins (i.e. Rep78 and Rep68) are required for AAV duplex DNA replication, but the small Rep proteins (i.e. Rep52 and Rep40) appear to be needed for progeny, single-strand DNA accumulation (Chejanovsky and Carter, 1989, Virology 173:120-128). Rep68 and Rep78 bind specifically to the hairpin conformation of the AAV ITR and possess several enzyme activities required for resolving replication at the AAV termini. Rep52 and Rep40 have none of these properties. Recent reports by C. Hxc3x6lscher et al. (1994, J. Virol. 68:7169-7177; and 1995, J. Virol. 69:6880-6885) suggest that expression of Rep 78 or Rep 68 may in some circumstances be sufficient for infectious particle formation.
The Rep proteins, primarily Rep78 and Rep68, also exhibit pleiotropic regulatory activities including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894; Labow et al., 1987, Mol. Cell. Biol., 7:1320-1325; Klileifet al., 1991, Virology, 181:738-741). The AAV p5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894). Due to the inhibitory effects of expression of rep on cell growth, constitutive expression of rep in cell lines has not been readily achieved. For example, Mendelson et al. (1988, Virology, 166:154-165) reported very low expression of some Rep proteins in certain cell lines after stable integration of AAV genomes.
The capsid proteins VP1, VP2, and VP3 share a common overlapping sequence, but VP1 and VP2 contain additional amino terminal sequences. All three proteins are encoded by the same cap gene reading frame typically expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 can be generated from this mRNA by use of alternate initiation codons. Generally, transcription from p40 yields a 2.6 kb precursor mRNA which can be spliced at alternative sites to yield two different transcripts of about 2.3 kb. VP2 and VP3 can be encoded by either transcript (using either of the two initiation sites), whereas VP1 is encoded by only one of the transcripts. VP3 is the major capsid protein, typically accounting for about 90% of total virion protein. VP1 is coded from a minor mRNA using a 3xe2x80x2 donor site that is 30 nucleotides upstream from the 3xe2x80x2 donor used for the major mRNA that encodes VP2 and VP3. All three proteins are required for effective capsid production. Mutations which eliminate all three proteins (Cap-negative) prevent accumulation of single-strand progeny AAV DNA, whereas mutations in the VP1 amino-terminus (xe2x80x9cLip-negativexe2x80x9d or xe2x80x9cInf-negativexe2x80x9d) can permit assembly of single-stranded DNA into particles but the infectious titer is greatly reduced.
The genetic analysis of AAV that was highlighted above was largely based upon mutational analysis of AAV genomes cloned into bacterial plasmids. In early work, molecular clones of infectious genomes of AAV were constructed by insertion of double-strand molecules of AAV into plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senpathy and Carter, 1984, J. Biol. Chem., 259:4661-4666). Transfection of such AAV recombinant plasmids into mammalian cells that were also infected with an appropriate helper virus, such as adenovirus, resulted in rescue and excision of the AAV genome free of any plasmid sequence, replication of the rescued genome and generation of progeny infectious AAV particles. This provided the basis for performing genetic analysis of AAV as summarized above and permitted construction of AAV transducing vectors.
Based on the genetic analysis, the general principles of AAV vector construction were defined as reviewed recently (Carter, 1992, Current Opinions in Biotechnology, 3:533-539; Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158:97-129). AAV vectors are generally constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with foreign DNA to generate a recombinant AAV (rAAV) vector or xe2x80x9cpro-vectorxe2x80x9d. In the vector, the terminal (ITR) portions of the AAV sequence must generally be retained intact because these regions are generally required in cis for several functions, including excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome. In some situations, providing a single ITR may be sufficient to carry out the functions normally associated with two wild-type ITRs (see, e.g., Samulski, R. J. et al., WO 94/13788, published Jun. 23, 1994). The vector can then be packaged into an AAV particle to generate an AAV transducing virus by transfection of the vector into cells that are infected by an appropriate helper virus such as adenovirus or herpesvirus; provided that, in order to achieve replication and encapsidation of the vector genome into AAV particles, the vector must be complemented for any AAV functions required in trans, particularly rep and cap, that were deleted in construction of the vector.
Such AAV vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents (reviewed in Carter, 1992, Current Opinion in Biotechnology, 3:533-539; Muzcyzka, 1992, Curr. Top. Microbiol. Immunol. 158:97-129 ) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of high-frequency transduction and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992a, Am. J. Respir. Cell Mol. Biol. 7:349-356; Egan et al., 1992, Nature, 358:581-584; Flotte et al., 1993a, J. Biol. Chem. 268:3781-3790; and Flotte et al., 1993b, Proc. Natl. Acad. Sci. USA, 93:10163-10167); human bone marrow-derived erythroleukemia cells (see, e.g., Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261); as well as brain, eye and muscle cells. AAV may not require active cell division for transduction and expression which would be another clear advantage over retroviruses, especially in tissues such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
There are at least two desirable features of any AAV vector designed for use in human gene therapy. First, the transducing vector must be generated at titers sufficiently high to be practicable as a delivery system. This is especially important for gene therapy stratagems aimed at in vivo delivery of the vector. For example, it is likely that for many desirable applications of AAV vectors, such as treatment of cystic fibrosis by direct in vivo delivery to the airway, the required dose of transducing vector may be in excess of 1010 particles. Secondly, the vector preparations are preferably essentially free of wild-type AAV virus (or any replication-competent AAV). The attainment of high titers of AAV vectors has been difficult for several reasons including preferential encapsidation of wild-type AAV genomes (if they are present or generated by recombination), and the difficulty in generating sufficient complementing functions such as those provided by the wild-type rep and cap genes. Useful cell lines expressing such complementing functions have been especially difficult to generate, in part because of pleiotropic inhibitory functions associated with the rep gene products. Thus, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express rep at very low levels.
The first AAV vectors described contained foreign reporter genes such as neo, cat or dhfr expressed from AAV transcription promoters or an SV40 promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081; Hernonat and Muzyczka, 1984, Proc. Natl. Acad. Sci. USA, 81:6466-6470; Tratschin et al., 1985, Mol. Cell. Biol. 5:3251-3260; McLaughlin et al., 1988, J. Virol, 62:1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol., 7:349-356). These vectors were packaged into AAV-transducing particles by co-transfection into adenovirus-infected cells together with a second xe2x80x9cpackaging plasmidxe2x80x9d containing the AAV rep and cap genes expressed from the wild-type AAV transcription promoters. Several strategies have been employed in attempts to prevent encapsidation of the packaging plasmid. In some cases, (Hermonat and Muzyczka, 1984; McLaughlin et al., 1988) a large region of bacteriophage lambda DNA was inserted into the packaging plasmid within the AAV sequence to generate an oversized genome that could not be packaged. In other cases, (Tratschin et al., 1984b; Tratschin et al., 1985, Lebkowski et al., 1988), the packaging plasmid had deleted the ITR regions of AAV so that it could not be excised and replicated and thus could not be packaged. All of these approaches failed to prevent generation of particles containing replication-competent AAV DNA and also failed to generate effective high titers of AAV transducing particles. Indeed, titers of not more than 104 infectious particles per ml were cited by Hermonat and Muzyczka, 1984.
In many studies, the presence of overlapping homology between AAV sequences present in the vector and packaging plasmids resulted in the production of replication-competent AAV particles. It was shown by Senapathy and Carter (1984, J. Biol. Chem. 259:4661-4666) that the degree of recombination in such a system is approximately equivalent to the degree of sequence overlap. It was suggested in a review of the early work (Carter 1989, Handbook of Parvoviruses, Vol. II, pp. 247-284, CRC Press, Boca Raton, Fla.) that titers of 106 infectious particles per ml might be obtained, but this was based on the above-cited studies in which large amounts of replication-competent AAV contaminated the vector preparation. Such vector preparations containing replication-competent AAV will generally not be preferred for human gene therapy. Furthermore, these early vectors exhibited low transduction efficiencies and did not transduce more than 1 or 2% of cells in cultures of various human cell lines even though the vectors were supplied at multiplicities of up to 50,000 particles per cell. This may have reflected in part the contamination with replication-competent AAV particles and the presence of the AAV rep gene in the vector. Furthermore, Samulski et al. (1989, J. Virol. 63:3822-3828) showed that the presence of wild-type AAV significantly enhanced the yield of packaged vector. Thus, in packaging systems where the production of wild-type AAV is eliminated, the yield of packaged vector may actually be decreased. Nevertheless, for use in any human clinical application it will be preferable to essentially eliminate production of replication-competent AAV.
Additional studies (McLaughlin et al., 1988; Lebkowski et al., 1988) attempting to generate AAV vectors lacking the AAV rep or cap genes still generated replication-competent AAV and still produced very low transduction frequencies on human cell lines. Thus, McLaughlin et al., 1988 reported that AAV rep-negative cap-negative vectors containing the neo gene packaged with the same packaging plasmid used earlier by Hermonat and Muzyczka (1984) still contained replication-competent AAV. As a consequence, it was only possible to use this virus at a multiplicity of 0.03 particles per cell (i.e., 300 infectious units per 10,000 cell) to avoid double hits with vector and wild-type particles. Thus, when 32,000 cells were infected with 1000 infectious units, an average of 800 geneticin-resistant colonies was obtained. Although this was interpreted as demonstrating that the virus was capable of yielding a transduction frequency of 80%, in fact only 2.5% of the cells were transduced. Thus the effectively useful titer of this vector was limited. Furthermore, this study did not demonstrate that the actual titer of the vector preparation was any higher than those obtained previously by Hermonat and Muzyczka (1984). Similarly, Lebkowski et al., 1988, packaged AAV vectors which did not contain either a rep or cap gene, using an ori-negative packaging plasmid (pBa1A) identical to that used earlier by Tratschin et al., (1984b, 1985), and reported transduction frequencies that were similarly low, in that for several human cell lines not more than 1% of the cells could be transduced to geneticin resistance even with their most concentrated vector stocks. Lebkowski et al., (1988) did not report the actual vector titers in a meaningful way but the biological assays, showing not more than 1% transduction frequency when 5xc3x97106 cells were exposed to three ml of vector preparation, indicate that the titer was less than 2xc3x97104 geneticin resistant units per ml. Also, the pBa1A packaging plasmid contains overlapping homology with the ITR sequence in the vector and can lead to generation of replication-competent AAV by homologous recombination.
Laface et al. (1988) used the same vector as that used by Hermonat and Muzyczka (1984) prepared in the same way and obtained a transduction frequency of 1.5% in murine bone marrow cultures, again showing very low titer.
Samulski et al. (1987, J. Virol., 61:3096-3101) constructed a plasmid called pSub201 which contained an intact AAV genome in a bacterial plasmid but which had a deletion of 13 nucleotides at the extremity of each ITR and thus was rescued and replicated less efficiently than other AAV plasmids that contained the entire AAV genome. Samulski et al. (1989, J. Virol., 63:3822-3828) constructed AAV vectors based on pSub201 but deleted for rep and cap and containing either a hyg or neo gene expressed from an SV40 early gene promoter. They packaged these vectors by co-transfection with a packaging plasmid called pAAV/Ad which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR. In this packaging plasmid the AAV rep and cap genes were expressed from their native AAV promoters (i.e. p5, p19 and p40, as discussed above). The function of the adenovirus ITR in pAAV/Ad was thought to enhance the expression level of AAV capsid proteins. However, rep is expressed from its homologous promoter and is negatively regulated and thus its expression is limited. Using their encapsidation system, Samulski et al. generated AAV vector stocks that were substantially free of replication-competent AAV but had transducing titers of only 3xc3x97104 hygromycin-resistant units per ml of supernatant. When a wild-type AAV genome was used in the packaging plasmid, the titer of the AAV vector prep was increased to 5xc3x97104 hygromycin-resistant units per ml. The low titer produced in this system thus appears to have been due in part to the defect in the ITR sequences of the basic pSub201 plasmid used for vector construction and in part due to limiting expression of AAV genes from pAAV/Ad. In an attempt to increase the titer of the AAVneo vector preparation, Samulski et al. generated vector stocks by transfecting, in bulk, thirty 10-cm dishes of 293 cells and concentrating the vector stock by banding in CsCl. This produced an AAVneo vector stock containing a total of 108 particles as measured by a DNA dot-blot hybridization assay. When this vector stock was used at multiplicities of up to 1,000 particles per cell, a transduction frequency of 70% was obtained. This suggests that the particle-to-transducing ratio is about 500 to 1,000 particles since at the ratio of one transducing unit per cell the expected proportion of cells that should be transduced is 63% according to the Poisson distribution.
Although the system of Samulski et al. (1989), using the vector plasmid pSub201 and the packaging plasmid pAAV/Ad, did not have overlapping AAV sequence homology between the two plasmids, there is overlapping homology at the XbaI sites and recombination of these sites can lead to the generation of complete replication-competent AAV. That is, although overlapping homology of AAV sequence is not present, the complete AAV sequence is contained within the two plasmids and the plasmids share a short (non-AAV) sequence that might facilitate recombination to generate replication-competent AAV, which is undesirable. That this class of recombination occurs in AAV plasmids was shown by Senapathy and Carter (1984, J. Biol. Chem. 259:466-4666). Given the problems of low titer, and the capability of generating wild-type recombinants, the system described by Samulski et al., 1989, does not have practical utility for human gene therapy.
Several other reports have described AAV vectors. For example, Srivastava et al., (1989, Proc. Natl. Acad. Sci. USA, 86:8078-8082) described an AAV vector based on the pSub201 plasmid of Samulski et al. (1987), in which the coding sequences of AAV were replaced with the coding sequences of another parvovirus, B19. This vector was packaged into AAV particles using the pAAV/Ad packaging plasmid to generate a functional vector, but titers were not reported. This system was based on pSub201 and thus suffers from the defect described above for this plasmid. Second, the vector and the packaging plasmid contained overlapping AAV sequences (the ITR regions) and thus recombination yielding contaminating wild-type virus is highly likely.
Chatterjee et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 85-89), Wong et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 183-189), and Chatterjee et al. (1992, Science, 258:1485-1488) describe AAV vectors designed to express antisense RNA directed against infectious viruses such as HIV or Herpes simplex virus. However, these authors did not report any titers of their AAV vector stocks. Furthermore, they packaged their vectors using an ori-negative packaging plasmid analogous to that used by Tratschin et al. (1984b, 1985) containing the BalA fragment of the AAV genome and therefore their packaging plasmid contained AAV vector sequences that have homology with AAV sequences that were present in their vector constructs. This will also lead to generation of replication-competent AAV. Thus, Chatterjee et al., and Wong et al., used a packaging system known to give only low titer and which can lead to generation of replication-competent AAV genomes because of the overlapping homology in the vector and packaging sequences.
Other reports have described the use of AAV vectors to express genes in human lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy, 11:231-237) or a human erythroid leukemia cell line (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261) with vectors based on the pSub201 vector plasmid and pAAV/Ad packaging plasmid. Again, titers of vector stocks were not reported and were apparently low because a selective marker gene was used to identify those cells that had been successfully transduced with the vector.
Transduction of human airway epithelia cells, grown in vitro from a cystic fibrosis patient, with an AAV vector expressing the selective marker gene neo from the AAV p5 promoter was reported (Flotte et al., 1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356). In this study the AAVneo vector was packaged into AAV particles using the pAAV/Ad packaging plasmid. Up to 70% of the cells in the culture could be transduced to geneticin resistance and the particle-to-transducing ratio was similar to that reported by Samulski et al. (1989). Thus to obtain transduction of 70% of the cells, a multiplicity of up to several hundred vector particles per cell was required. Transduction of human airway epithelial cells in in vitro culture using an AAV transducing vector that expressed the cystic fibrosis transmembrane conductance regulator (CFTR) gene from the AAV ITR promoter showed that the cells could be functionally corrected for the electrophysiological defect in chloride channel function that exists in cells from cystic fibrosis patients (Egan et al., Nature, 1992, 358:581-584; Flotte et al., J. Biol. Chem.268:3781-3790).
The above-cited studies suggest that AAV vectors have potential utility as vectors for treatment of human disease by gene therapy. However, the difficulty in generating sufficient amounts of AAV vectors has been a severe limitation on the development of human gene therapy using AAV vectors. One aspect of this limitation is that there have been very few studies using AAV vectors in in vivo animal models (see, e.g., Flotte et al., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154). This is generally a reflection of the difficulty associated with generating sufficient amounts of AAV vector stocks having a high enough titer to be useful in analyzing in vivo delivery and gene expression.
One of the limiting factors for AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used. In the absence of suitable cell lines expressing sufficient levels of the AAV trans complementing functions, such as rep and cap, packaging of AAV vectors has been achieved in adenovirus-infected cells by co-transfection of a packaging plasmid and a vector. The efficiency of this process is expected to be limited by the efficiency of transfection of each of the plasmid constructs, and by the low level of expression of Rep proteins from the packaging plasmids described to date. Each of these problems appears to relate to the biological activities of the AAV Rep proteins which are known to be associated with pleiotropic inhibitory effects. In addition, as noted above, all of the packaging systems described above have the ability to generate replication-competent AAV by recombination.
The difficulty in generating cell lines stably expressing functional Rep apparently reflects a cytotoxic or cytostatic function of Rep as shown by the inhibition, by Rep protein, of neo-resistant colony formation (Labow et al., 1987; Trempe et al., 1991). This also appears to relate to the tendency of Rep to reverse the immortalized phenotype in cultured cells, which has made the production of cell lines stably expressing functional rep extremely difficult. Several attempts to generate cell lines expressing rep have been made. Mendelson et al., (1988, Virology, 166:154-165) reported obtaining in one cell line some low level expression of AAV Rep52 protein but no Rep78 or Rep68 protein after stable transfection of Hela or 293 cells with plasmids containing an AAV rep gene. Because of the absence of Rep78 and Rep68 proteins, vector could not be produced in the cell line. Another cell line made a barely detectable amount of Rep78 which was nonfunctional.
Vincent et al. (1990, Vaccines 90, Cold Spring Harbor Laboratory Press, pp. 353-359) attempted to generate cell lines containing the AAV rep and cap genes expressed from the normal AAV promoters, but these attempts were not successful either because the vectors were contaminated with a 100-fold excess of wild-type AAV particles or because the vectors were produced at only very low titers of less than 4xc3x97103 infectious particles.
Other variations that have been proposed include systems based on the production of AAV Cap proteins that might be used to reconstitute AAV particles, e.g. by assembly in vitro (see, e.g., WO 96/00587, published Nov. 1, 1996); systems employing AAV rep-cap genes on a helper virus (see, e.g., WO 95/06743, published Mar. 9, 1995); and systems employing helper viruses from non-human mammals (see, e.g., WO 95/20671, published Aug. 3, 1995).
In yet another approach, Lebkowski et al. (U.S. Pat. No. 5,173,414, issued Dec. 22, 1992) constructed cell lines containing AAV vectors in an episomal plasmid. These cell lines could then be infected with adenovirus and transfected with the trans-complementing AAV functions rep and cap to generate preparations of AAV vector. It is claimed that this allows higher titers of AAV stocks to be produced. However, in the examples shown, the only information relative to titer that is shown is that one human cell line, K562, could be transduced at efficiencies of only 1% or less, which does not indicate high titer production of any AAV vector. In this system the vector is carried as an episomal (unintegrated) construct, and it is stated that integrated copies of the vector are not preferred. In a subsequent patent (U.S. Pat. No. 5,354,678, issued Oct. 11, 1994), Lebkowski et al. introduce rep and cap genes into the cell genome but the method again requires the use of episomal AAV transducing vectors comprising an Epstein-Barr virus nuclear antigen (EBNA) gene and an Epstein-Barr virus latent origin of replication; and, again, the only information relative to titer indicated that it was fairly low.
The approach to packaging of rAAV vectors described by Lebkowski et al., 1992, can be undesirable in several ways. First, maintaining the rAAV vector as an unintegrated, high copy number episomal plasmid in a cell line is not desirable because the copy number per cell cannot be rigorously controlled and episomal DNA is much more likely to undergo rearrangement leading to production of defective vectors. Secondly, in this system, the vector must still be packaged by infecting the cell line with adenovirus and introducing a plasmid containing the AAV rep and cap genes. The plasmid used by Lebkowski et al. (1992) was again pBa1A, which has overlapping homology with the vector ITR sequences and can result in generation of replication-competent AAV. Third, in the pBa1A packaging plasmid used by Lebkowski et al., 1988, 1992, the rep gene is expressed from its homologous p5 promoter and, since rep is generally negatively autoregulated, this would tend to limit rep expression.
The problem of suboptimal levels of rep expression after plasmid transfection thus also relates to another biological activity of these proteins. There is evidence (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894) that AAV Rep proteins down-regulate their own expression from the AAV-p5 promoter which has been used in the various previously described packaging constructs such as pAAV/Ad (Samulski et al., 1989) or pBa1A (Lebkowski et al., 1988, 1992).
Another attempt to develop cell lines expressing functional rep activity was recently published by Hxc3x6lscher et al. (1994, J. Virol. 68:7169-7177). They described the generation of cell lines in which rep was placed under control of a glucocorticoid-responsive MMTV promoter. Although they observed particle formation, the particles were apparently noninfectious. Additional experiments indicated that the defect was quite fundamental; namely, there was virtually no accumulation of single-stranded rAAV DNA in the cells. Production of infectious particles required an additional transient transfection with constitutive highly-expressed rep constructs (i.e. they had to xe2x80x9cadd backxe2x80x9d Rep activity to cells that were supposed to be able to provide it themselves). Several other approaches to generating AAV packaging cell lines have also been described recently, see, e.g., T. Flotte et al., WO 95/13365 (Targeted Genetics Corporation and Johns Hopkins University); J. Trempe et al., WO 95/13392 (Medical College of Ohio); and J. Allen, WO 96/17947 (Targeted Genetics Corporation).
There is a significant need for methods that can be used to efficiently generate rAAV vectors that are essentially free of wild-type or other replication-competent AAV; and a corresponding need for cell lines that can be used to effectively generate such rAAV vectors.
One of the basic challenges for gene therapy has been the development of strategies for transduction of cells and tissues which cannot be easily manipulated ex vivo or which are not actively dividing. AAV vectors can achieve in vivo gene transfer in the respiratory tract, for example, but high titers are critical so as to allow for the delivery of sufficiently high multiplicity of vector in as small a volume as possible. In addition, prior art techniques that do not substantially eliminate the generation of replication-competent AAV (xe2x80x9crcAxe2x80x9d) are generally unsatisfactory. In addition, stable, AAV packaging cell lines have been elusive, mainly due to the activities of Rep proteins, which tend to down-regulate their own expression and can negatively affect the lost cell. These concerns, particularly when considered in combination, make packaging methodology of central importance in AAV-based gene therapy applications. Although a number of suggestions have been proposed in the art, very few of them have actually been tested, and many that have are practically inadequate for reasons as dicussed above.
The closely-coupled and tightly-regulated rep and cap functions characteristic of wild-type AAV, and various prior packaging systems, are uncoupled and reorganized in the packaging cell lines of the present invention. The inventors have found that their redesigned system is capable of providing significantly greater quantities of heat-stable recombinant AAV vectors, while at the same time greatly reducing the possibility of generating replication-competent AAV particles.
1. A mammalian cell useful for high efficiency packaging of a recombinant adeno-associated virus (rAAV) vector, said cell comprising at least one copy of each of the following AAV split-packaging genes:
(i) an AAV split-cap gene, wherein said split-cap gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said split-cap gene is operably linked to a heterologous promoter; and
(ii) an AAV rep78 gene, wherein said rep78 gene is uncoupled from Cap-specific sequences of an AAV cap gene, and wherein said rep78 gene is operably linked to a heterologous promoter. Preferably, the rep78 gene is operably linked to an inducible promoter; the split-cap gene can be operably linked to an inducible promoter or to a constitutive promoter.
Important properties of such cells include the fact that they can generate high titers of infectious rAAV particles, as illustrated below, and they have a greatly reduced likelihood of generating replication-competent AAV. Indeed, the frequency of generating rcA is expected to be less than 1 in 108 particles generated, and is likely to be much less frequent than that (as described below).
2. A mammalian cell according to embodiment 1, further comprising an AAV rep52 gene, wherein said rep52 gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said rep52 gene is operably linked to a promoter, preferably a heterologous promoter. The use of both split-cap and rep52 genes has been found to result in the generation of substantially increased amounts of infectious rAAV particles, and the resulting particles are heat stable to at least 56 degrees Celsius for one hour, even when packaging an xe2x80x9cover-sizedxe2x80x9d rAAV vector (i.e. larger than the normal AAV genome size) such as the CFTR vector exemplified below.
3. A mammalian cell according to one of the preceding embodiments, wherein at least one of said promoters is an inducible heterologous promoter; preferably at least the rep78 promoter is an inducible heterologous promoter; most preferably, it is a helper-virus-inducible promoter. The rep52 and split-cap genes can also be linked to heterologous promoters, including helper-virus-inducible promoters.
4. A mammalian cell according to one of the preceding embodiments, wherein at least one of said AAV split-packaging genes is operably linked to a heterologous enhancer; preferably, at least two of said AAV split-packaging genes, particularly the split-cap and rep52 genes, are operably linked to heterologous enhancers.
5. A mammalian cell according to one of the preceding embodiments, wherein at least one of said AAV split-packaging genes is stably integrated into said cell, in one or multiple copies. A preferred embodiment comprises a stably-integrated rep78 gene operably linked to a helper-virus-inducible promoter, and multiple copies of the split-cap and rep52 genes, stably-integrated or transiently introduced.
6. A mammalian cell according to one of the preceding embodiments, wherein at least two different AAV split-packaging genes are stably integrated into said cell, in one or multiple copies.
7. A mammalian cell according to one of the preceding embodiments, wherein at least three different AAV split-packaging genes are stably integrated into said cell, in one or multiple copies.
8. A mammalian cell according to one of the preceding embodiments, further comprising a recombinant AAV vector; preferably, said recombinant AAV vector comprises two AAV inverted terminal repeats and a heterologous gene of interest operably linked to a promoter.
9. A mammalian cell according to embodiment 8, wherein said heterologous gene of interest is a therapeutic gene.
10. A mammalian cell according to embodiment 9, wherein said therapeutic gene encodes a cystic fibrosis transmembrane regulator.
11. A mammalian cell according to one of the preceding embodiments, wherein said mammalian cell is capable of producing at least about 100 recombinant AAV particles per cell. As is described below, virus production is generally initiated by infecting the cells with a helper virus such as adenovirus, or by providing an alternative source of helper virus functions.
12. A mammalian cell according to one of the preceding embodiments, wherein said mammalian cell is capable of producing at least about 200 recombinant AAV particles per cell.
13. A mammalian cell according to one of the preceding embodiments, wherein said cell is capable of producing at least about 400 recombinant AAV particles per cell.
14. A mammalian cell according to one of the preceding embodiments, wherein said mammalian cell is a human cell. In preferred embodiments of the present invention, the cells are capable of producing high titers of heat-stable AAV particles (stable to heating at 56 degrees C. for 30 minutes without substantial loss of infectivity). Also, as discussed in more detail below, preferred split-packaging cells also exhibit a greatly reduced tendency to generate replication-competent AAV (xe2x80x9crcAxe2x80x9d) particles. In preferred cells, the frequency of generation of rcA is less than about 1 per 106 particles produced, more preferably less that about 1 per 108, still more preferably less than about 1 per 1010, most preferably less than about 1 per 1012.
15. A polynucleotide expression vector useful in preparing an AAV split-packaging cell, said expression vector comprising a selectable marker and at least one AAV split-packaging gene selected from the group consisting of:
(i) an AAV split-cap gene, wherein said split-cap gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said split-cap gene is operably linked to a heterologous promoter, and preferably a heterologous enhancer;
(ii) an AAV rep78 gene, wherein said rep78 gene is uncoupled from Cap-specific sequences of an AAV cap gene, and wherein said rep78 gene is operably linked to a heterologous inducible promoter, preferably a heterologous helper-virus-inducible promoter; and
(iii) an AAV rep52 gene, wherein said rep52 gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said rep52 gene is operably linked to a heterologous promoter, preferably a heterologous helper-virus-inducible promoter.
16. A polynucleotide expression vector according to embodiment 15, comprising an AAV split-cap gene; preferably said gene is operably linked to a heterologous promoter.
17. A polynucleotide expression vector according to embodiment 15, comprising an AAV rep78 gene; preferably said gene is operably linked to a heterologous promoter, more preferably a helper-virus-inducible promoter.
18. A polynucleotide expression vector according to embodiment 15, comprising an AAV rep52 gene; preferably said gene is operably linked to a heterologous promoter.
19. A polynucleotide expression vector according to one of embodiments 15-18, wherein said expression vector comprises the following two AAV split-packaging genes:
(i) an AAV split-cap gene, wherein said split-cap gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said split-cap gene is operably linked to a heterologous promoter; and
(ii) an AAV rep52 gene, wherein said rep52 gene is uncoupled from Rep78-specific sequences of an AAV rep gene, and wherein said rep52 gene is operably linked to a heterologous promoter.
20. A polynucleotide expression vector according to embodiment 19, wherein said AAV split-cap gene and said AAV rep52 gene are arranged in tandem transcriptional orientation.
21. A polynucleotide expression vector according to embodiment 19, wherein said AAV split-cap gene and said AAV rep52 gene are arranged in divergent transcriptional orientation.
22. A method of generating a mammalian cell useful for high efficiency packaging of a recombinant adeno-associated virus (rAAV) vector, comprising introducing into said cell a polynucleotide expression vector according to one of embodiments 15-21.
23. A method of generating a mammalian cell useful for high efficiency packaging of an rAAV vector, comprising introducing into said cell at least two different polynucleotide expression vectors according to one of embodiments 15-21.
24. A method of generating a mammalian cell useful for high efficiency packaging of an rAAV vector, comprising introducing into said cell at least three different polynucleotide expression vectors according to one of embodiments 15-21.
25. A method of generating a mammalian cell useful for high efficiency packaging of an rAAV vector, comprising stably introducing into said cell a polynucleotide expression vector according to one of embodiments 15-21.
26. A method of generating a mammalian cell useful for high efficiency packaging of an rAAV vector, comprising stably introducing into said cell at least two different polynucleotide expression vectors according to one of embodiments 15-21.
27. A method of generating a mammalian cell useful for high efficiency packaging of an rAAV vector, comprising stably introducing into said cell at least three different polynucleotide expression vectors according to one of embodiments 15-21.
28. A mammalian cell generated according to one of embodiments 22-27.
29. A method of generating an infectious replication-incompetent rAAV vector, comprising introducing helper virus functions into a cell of claim 28, and incubating said cell.